In the coming year we shall extend our investigations of respiration-coupled H ion transport across the mitochondrial membrane by identifying the specific sites and carriers in the respiratory chain that furnish the energy for the H ion movements. With this information we propose to go further and examine the molecular mechanisms by which the protoms are transported, whether by loop mechanisms or by conformational changes of "proton-pumping" molecules, and how these mechanisms are related to the process of oxidative phosphorylation. Secondly, we propose to analyze further the mechanisms of respiration-dependent Ca 2 ion transport into and out of mitochondria, to establish more firmly the difference between the influx and efflux mechanisms, and, most important, to establish the nature of the metabolic conditions that regulate the relative rates of Ca 2 ion influx and efflux during steady-state respiration. Our third major objective is to work out the mechanism of enzymatic synthesis of phosphocitrate, which we have recently isolated from mitochondria and synthesized, as well as the mechanism of its enzymatic degradation. Further, we propose to examine the physical-chemical and biochemical mechanisms by which this substance apparently serves as a regulator of the transition of amorphous calcium phosphate into crystalline hydroxyapatite in the mitochondrial matrix, as well as in calcifying tissues.